Hironari Takaishi

Cutting Edge: TNF-α-Converting Enzyme (TACE/ADAM17) Inactivation in Mouse Myeloid Cells Prevents Lethality from Endotoxin Shock

TNF-α, a potent proinflammatory cytokine, is synthesized as a membrane-anchored precursor and proteolytically released from cells. Soluble TNF is the primary mediator of pathologies such as rheumatoid arthritis, Crohn’s disease, and endotoxin shock. The TNF-α converting enzyme (TACE), a disintegrin and metalloprotease 17 (ADAM17), has emerged as the best candidate TNF sheddase, but other proteinases can also release TNF. Because TACE-deficient mice die shortly after birth, we generated conditional TACE-deficient mice to address whether TACE is the relevant sheddase for TNF in adult mice. In this study, we report that TACE inactivation in myeloid cells or temporal inactivation at 6 wk offers strong protection from endotoxin shock lethality in mice by preventing increased TNF serum levels. These findings corroborate that TACE is the major endotoxin-stimulated TNF sheddase in mouse myeloid cells in vivo, thereby further validating TACE as a principal target for the treatment of TNF-dependent pathologies.

Long-term results of expansive open-door laminoplasty for cervical myelopathy--average 14-year follow-up study.

Study Design. Retrospective case series on long-term follow-up results of original expansive open-door laminoplasty for cervical myelopathy due to cervical spondylosis (CSM) and ossification of posterior longitudinal ligament (OPLL). Objectives. To elucidate efficacy and problems of original open-door laminoplasty to improve future surgical outcomes. Summary of Background Data. Little information is available on long-term outcomes of original open-door laminoplasty without grafts, implants, or instruments. Method. The study group included 80 patients who underwent original open-door laminoplasty and were followed for minimum 10 years. Clinical results, including Japanese Orthopedic Association scores, recovery rates, occurrences of complications, and long-term deterioration were investigated. Cervical alignments, type of OPLL, cervical range of motion, anteroposterior diameter of spinal canal, and progression of OPLL were assessed on plain radiographs. Spinal cord decompression was verified on magnetic resonance imaging. Results. Average Japanese Orthopedic Association score and recovery rate improved significantly until 3 years after surgery and remained at an acceptable level in both cervical spondylosis and OPLL patients with slight deterioration after 5 years. Segmental motor palsy developed in 8 patients. Late deterioration, mainly lower extremity motor score decline, developed in 8 CSM and 16 OPLL patients. Overall cervical range of motion decreased by 36%. Patients with cervical lordosis decreased gradually in both patient groups. Such changes in alignments did not affect surgical results in CSM patients, while OPLL patients with preoperative kyphosis had lower recovery rates than those with straight and lordotic alignments. OPLL progression that was detected in 66% of patients did not affect clinical results. Although infrequent, magnetic resonance imaging revealed atrophy of spinal cord, spinal cord compression at adjacent segments due to degenerative changes and OPLL progression. Conclusions. Long-term results of open-door laminoplasty without bone graft, graft substitutes, or instruments were satisfactory. However, segmental motor paralysis, kyphosis, established before and after surgery, OPLL progression, and late deterioration due to age-related degeneration remain challenging problems.

Joint Diseases and Matrix Metalloproteinases: A Role for MMP-13

The role of matrix metalloproteinases in disease has been investigated over the last two decades. A focus on this family of proteases is particularly emphasized in two major arthritides in humans, osteoarthritis and rheumatoid arthritis. Early work described the presence of multiple MMP family members in the joint of the disease state and recent advances in the development of new knockout mice and disease models have allowed investigators to directly test the role of the MMP proteases in arthritis. MMP-13 is expressed by chondrocytes and synovial cells in human OA and RA and is thought to play a critical role in cartilage destruction. The recent development of an MMP-13 knockout mouse has documented the important role for this enzyme in cartilage formation and further studies under disease conditions promise to reveal the function of this enzyme in disease pathology. This review describes a body of research that supports the development of novel selective MMP-13 inhibitors with the hope of developing these compounds in clinical trials for the treatment of arthritis.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification

Chondrocyte hypertrophy during endochondral ossification is a well-controlled process in which proliferating chondrocytes stop proliferating and differentiate into hypertrophic chondrocytes, which then undergo apoptosis. Chondrocyte hypertrophy induces angiogenesis and mineralization. This step is crucial for the longitudinal growth and development of long bones, but what triggers the process is unknown. Reactive oxygen species (ROS) have been implicated in cellular damage; however, the physiological role of ROS in chondrogenesis is not well characterized. We demonstrate that increasing ROS levels induce chondrocyte hypertrophy. Elevated ROS levels are detected in hypertrophic chondrocytes. In vivo and in vitro treatment with N-acetyl cysteine, which enhances endogenous antioxidant levels and protects cells from oxidative stress, inhibits chondrocyte hypertrophy. In ataxia telangiectasia mutated (Atm)–deficient (Atm−/−) mice, ROS levels were elevated in chondrocytes of growth plates, accompanied by a proliferation defect and stimulation of chondrocyte hypertrophy. Decreased proliferation and excessive hypertrophy in Atm−/− mice were also rescued by antioxidant treatment. These findings indicate that ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes.

Macrophage- and neutrophil-dominant arthritis in human IL-1α transgenic mice

To study the effects of IL-1α in arthritis, we generated human IL-1α (hIL-1α). Transgenic mice expressed hIL-1α mRNA in various organs, had high serum levels of hIL-1α, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1α. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1α was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNs. Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.

Surgical treatment of intramedullary spinal cord tumors: prognosis and complications

Study design:Retrospective case series.Objective:To evaluate our recent treatment strategy for intramedullary spinal cord tumors.Setting:Department of Orthopaedic Surgery, Keio University, Japan.Methods:We reviewed 68 cases of intramedullary tumors (ependymoma, 33; astrocytoma, 23; hemangioblastoma, 12), treated surgically between 1994 and 2003. There were 42 males and 26 females whose mean age at the time of surgery was 43 years. The mean follow-up period was 6.2 years. The tumor malignancy grade according to the WHO classification was astrocytoma grade I, 3; grade II, 8 (low-grade: 11 cases); grade III, 10; grade IV, 2 (high-grade: 12 cases). All ependymomas were grade II. Three of the 12 hemangioblastomas were associated with von Hippel–Lindau disease.Results:Total excision was achieved in 90% of the ependymomas and functional improvement was obtained when the preoperative neurological deficit was mild. Approximately 50% of low-grade astrocytomas could be totally excised with favorable survival outcomes, suggesting that total excision should be attempted for low-grade astrocytomas. However, total excision of high-grade tumors was difficult and the functional outcomes were poor. Cordotomy should be considered in patients with a thoracic high-grade astrocytoma. Total resection was possible in 92% of hemangioblastoma, and the functional outcomes were good, however, more attention should be paid for tumors with feeding arteries on the ventral side and for those associated with von Hippel–Lindau disease.Conclusions:Predictors of good surgical outcome for intramedullary spinal cord tumors were histological grades of the tumors, surgical margins, and neurological status of the patient before surgery.

Matrix metalloproteinase-3 production by human degenerated intervertebral disc

To assess its possible role in the pathophysiology of intervertebral disc degeneration, we investigated the production of matrix metalloproteinase-3 (MMP-3) using human intervertebral disc explant culture. Five normal and 10 degenerated disc specimens were used. The levels of MMP-3 released in the medium were measured with use of an enzyme immunoassay. The results showed that the level of MMP-3 in the degenerated group (0.57 microg/ml/mg wet weight; n = 10) was significantly higher than that of the control group (0.29 microg/ml/mg wet weight; n = 5) (p < 0.05). Immunostaining of MMP-3 revealed that the ratio of positive staining cells in the degenerated group was greater than that of the control group. These observations suggest that MMP-3 produced by human intervertebral disc may be involved in the intervertebral disc degeneration, particularly in the initiation of matrix degradation of intervertebral disc.

Conditional inactivation of TACE by a Sox9 promoter leads to osteoporosis and increased granulopoiesis via dysregulation of IL-17 and G-CSF

The TNF-α converting enzyme (TACE/ADAM17) is involved in the proteolytic release of the ectodomain of diverse cell surface proteins with critical roles in development, immunity, and hematopoiesis. As the perinatal lethality of TACE-deficient mice has prevented an analysis of the roles of TACE in adult animals, we generated mice in which floxed Tace alleles were deleted by Cre recombinase driven by a Sox9 promoter. These mutant mice survived up to 9–10 mo, but exhibited severe growth retardation as well as skin defects and infertility. The analysis of the skeletal system revealed shorter long bones and prominent bone loss, characterized by an increase in osteoclast and osteoblast activity. In addition, these mice exhibited hypercellularity in the bone marrow and extramedullary hematopoiesis in the spleen and liver. Flow cytometric analysis of the bone marrow cells showed a sharp increase in granulopoiesis and in the population of c-Kit-1+ Sca-1+ lineage− cells, and a decrease in lymphopoiesis. Moreover, we found that serum levels of IL-17 and G-CSF were significantly elevated compared with control littermates. These findings indicate that TACE is associated with a regulation of IL-17 and G-CSF expression in vivo, and that the dysregulation in G-CSF production is causally related to both the osteoporosis-like phenotype and the defects in the hematopoietic system.

IL-27 Abrogates Receptor Activator of NF-κB Ligand-Mediated Osteoclastogenesis of Human Granulocyte-Macrophage Colony-Forming Unit Cells through STAT1-Dependent Inhibition of c-Fos

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Rα (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-κB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-κB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.